ESEARCH ARTICLE R ScienceAsia 41 (2015): 79–86 doi: 10.2306/scienceasia1513-1874.2015.41.079 Is major (Zoll.) Miquel a big H. ovalis (R. Brown) J.D. Hooker? An evaluation based on age, morphology, and ITS sequence

a b c c, Piyalap Tuntiprapas , Satoshi Shimada , Supattra Pongparadon , Anchana Prathep ∗ a Excellence Centre for Biodiversity of Peninsular Thailand, Faculty of Science, Prince of Songkla University, Thailand b Graduate School of the Natural/Applied Sciences, Division Ochanomizu University, Tokyo c Seaweed and Research Unit, Department of Biology, Faculty of Science, Prince of Songkla University, HatYai, Songkhla 90112 Thailand

∗Corresponding author, e-mail: [email protected] Received 26 Dec 2014 Accepted 1 Mar 2015

ABSTRACT: The common seagrass, (R. Brown) J.D. Hooker, is highly variable morphologically. It adapts well to various environmental conditions rendering the various forms unclear taxonomically. Halophila species were collected along the coast of southern Thailand. The morphology was quantified according to different parts of the leaf and the ages of leaves. Some samples had significantly different characters from H. ovalis: the lengths of their leaves ranged from 11.7–29.4 mm, and the widths from 5.6–14.8 mm; there were 9–18 cross veins. Phylogenetic analyses based on ribosomal internal transcribed spacer sequences divided them into two groups: one agrees with H. ovalis and the other with H. major. We suggest that leaf size at maturity (age iii-iv) and the ½ ratio between the leaf width and the space between the intra-marginal vein and lamina margin are important characters that distinguish Halophila species.

KEYWORDS: seagrass, nrITS, leaf age, Thailand

INTRODUCTION and taxonomic studies have not been revised in the last 10 years. For example, only H. decipiens, The Halophila Thouars (1806), family Hy- H. ovalis, and H. ovata have been reported in the drocharitaceae (, Monocots) 1 has a Flora of Thailand 17, where H. ovata was placed broad global distribution 2, and is one of the most as a synonym of H. minor. Later however it was important marine due to its ecological roles recognized as two distinct species 18. H. ovata is as primary producer in marine environments 3. now an illegitimate name and is proposed as H. gau- Halophila ovalis (R. Brown) J.D. Hooker is the most dichaudii 19; but a recent study 12 suggested that common species in this genus found in the Indo- H. gaudichaudii was a synonym of H. nipponica. Pacific, the temperate North Pacific, the temperate These observations reveal that the taxonomic status Southern Oceans, and has recently been observed of the group is still unclear. Besides, molecular in the Tropical Atlantic Ocean 4. It is well known for studies by Uchimura et al 12 revealed that H. major its variable morphology and adaptability to various occurred in Thailand, which was the first record of environmental conditions 5–8. Although H. ovalis H. major in Thailand. A recent report by Nguyen is widely distributed, it has been represented as a et al 20 suggested the occurrence of H. major in single collective species 1,9 . Thailand; however its morphological features had The five species of Halophila reported from never been examined. Thailand are H. ovalis, H. beccarii, H. minor, H. de- During our recent surveys, we have found many cipiens, and H. major 10–12. H. ovalis is also common Halophila specimens in several locations with sim- in Thai waters, forming extensive beds along the An- ilar characters to H. ovalis, but some with greater daman coast. It is well documented as food for the leaf size and thick leaf. Both forms grow in the , an endangered marine mammal 11, 13, 14. subtidal zones also mixed with Thalassia hemprichii Seagrass studies in Thailand are however scant 15, 16 and Cymodocea serrulata. Recent studies using var-

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mated sequencing. The following pair of primers

BURMA was used for PCR and cycle-sequencing reactions: LAOS Ko Samui ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 THAILAND 0 0 Surathani Ko Tan Province CAMBODIA (50-TCCTCCGCTTATTGATATGC-30). PCR amplifica- Gulf of Ban Pak Meang Thailand tion was run on a PROGRAM TEMP CONTROL SYS- Andaman Ao Tueng Krabi Sea Ao Nang Province TEM (Astec, Fukuoka, Japan) and the profile of the Trang Province Leam Yong Lum reactions was an initial denaturation 1 min at 94 °C Trang Ko Muk Satun followed by 35 cycles of denaturation 45 s at 94 °C, Province Sampling Site Ko Libong 0 50 100 150 the primers annealing 45 s at 50 °C, and extension km 60 s at 72 °C, terminated by a final hold at 4 °C. Ko Lidee Yai

Ko Lao Leang The presence of the PCR-amplified products was Ko Lidee Lek Malaysia verified by agarose gel electrophoresis, followed by staining with ethidium bromide. Prior to cycle- sequencing, PCR-amplified products were cleaned Fig. 1 Field collection sites along the coastal southern using the QIAquick PCR Purification Kit (QIAGEN, Thailand. Valencia, CA, USA) and directly sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Kit ver. 3.1 (Applied Biosystems, CA, USA) according ious genetic markers of plastid sequences have clar- to the manufacturers. Cycle-sequencing reactions ified the identification of the Halophila species 21, 22. consisted of an initial step of 96 °C for 10 s, followed Ribosomal internal transcribed spacer (ITS) se- by 25 cycles (96 °C for 10 s, 50 °C for 5 s, 60 °C for quences could be used to study and identify the 4 min) and a final hold at 4 °C. Only the forward genetic relation between Halophila closely related strand was sequenced using a DNA autosequencer species 12, 20, 23, 24. It is unknown weather there is (ABI PRISM, 3130 Genetic Analyser, Applied Biosys- any difference in ITS molecular analysis between tems, CA, USA). big and small leaf morphological forms of H. ovalis. The sequences were aligned using CLUSTAL Thus this study evaluates the taxonomic status of X 25. Identical sequences within each species this large Halophila sp. by analysing the nuclear were excluded from the alignment. Additional ribosomal internal transcribed spacer (nrITS) se- 29 ingroup sequences were loaded from GenBank quences and measuring different leaf parts at var- (Table 1). H. decipiens Ostenfeld (AF366412) and ious ages. H. stipulacea (Forssk.) Asch. (AF366436) desig- nated as outgroups. Phylogenetic analysis were MATERIALS AND METHODS implemented using maximum likelihood (ML) and were collected from the intertidal and Bayesian Inference (BI). Prior to ML and BI analysis, subtidal zones along the coastal line in southern the best-fit model of nucleotide substitution was Thailand (Fig. 1). Samples were collected by walk- selected using the JMODELTEST 2.1.1 tool 26. The ing survey during low tide at intertidal zone area ML tree was constructed using RAXML 27 with the and by using SCUBA diving or snorkelling at the sub- HKY+I+G model. Support for branches was ob- tidal area. At each sampling point containing tained from 1000 bootstrap replications. BI analysis leaf, root, and rhizome having at least 3–4 leaf was performed using MRBAYES v.3.2.1 28, with a pairs were selected, cleaned, and preserved as dried random starting tree run for 5 000 000 generations, herbarium specimens. New and/or young leaves sampling tree every 1000 generations and a with a of Halophila with no epiphytes were preserved in burning of 5000 trees. silica gel for molecular studies. These vouchers The herbarium specimens were closely exam- herbarium specimens were deposited at Princess ined under a stereo microscope (Olympus SZX 12) Maha Chakri Sirindhorn Natural History Museum, and photographed using an Olympus DP 71. Each Prince of Songkla University, Hat Yai. leaf was divided into 4 equal sections from the Total DNA was extracted from 38 samples base to the apex (Fig. 2). The leaf morphological (Table 1) using the DNeasy Plant Mini Kit (QIA- characters were quantified as follows: leaf length GEN, Valencia, CA, USA) following the protocol of (LL) and leaf width (LW) in each of the four sec- the manufacturer. The nuclear ribosomal internal tions, number of cross veins (CV), counted from the transcribed spacer (nrITS) region including the 5.8S base of all cross vein (secondary vein) which are gene was selected for PCR amplification and auto- connected with the mid rib (primary vein), cross www.scienceasia.org ScienceAsia 41 (2015) 81

Table 1 Samples collected from southern Thailand and sequence data that use in this study. No. Taxon Location Voucher No. GenBank No. Source 1 H. ovalis Okinawa, Japan AB243973 23 2 H. ovalis Okinawa, Japan AB243975 23 3 H. ovalis Trang, Thailand AB436938 12 4 H. ovalis Trang, Thailand AB436939 12 5 H. ovalis Queensland, Australia AF366431 29 6 H. ovalis Marakanam, India KF620355 20 7 H. ovalis Kanyakumari, India KF620353 20 8 H. ovalis Trang, Thailand KF620350 20 9 H. ovalis Nakhon Si Thammarat, Thailand KF620345 20 10 H. ovalis Satun, Thailand KF620347 20 11 H. ovalis Lantau Island, Hong Kong KF620337 20 12 H. ovalis Cu Mong Lagoon, Vietnam KC175909 20 13 H. ovalis Sarawak, Malaysia KF620338 20 14 H. ovalis Tiga Island, Malaysia KF620339 20 15 H. ovalis Johore, Malaysia KF620346 20 16 H. ovalis Flores Island, Indonesia AB436930 12 17 H. ovalis Leam Yong Lum, Trang, Thailand PT 45.1.1* KP408228 † (Group Ho.1) 18 H. ovalis Leam Yong Lum, Trang, Thailand PT 45.2.1 KP408229 † (Group Ho.1) 19 H. ovalis Leam Yong Lum, Trang, Thailand PT 49.1.1* KP408230 † (Group Ho.1) 20 H. ovalis Leam Yong Lum, Trang, Thailand PT 49.2.2 KP408231 † (Group Ho.1) 21 H. ovalis Leam Yong Lum, Trang, Thailand PT 49.3.4 KP408232 † (Group Ho.1) 22 H. ovalis Leam Yong Lum, Trang, Thailand PT 52.1.2 KP408233 † (Group Ho.1) 23 H. ovalis Leam Yong Lum, Trang, Thailand CK 13-1 KP408234 † (Group Ho.1) 24 H. ovalis Leam Yong Lum, Trang, Thailand CK 14-2 KP408235 † (Group Ho.1) 25 H. ovalis Leam Yong Lum, Trang, Thailand CK 14-6 KP408236 † (Group Ho.1) 26 H. ovalis Leam Yong Lum, Trang, Thailand CK 14-7 KP408237 † (Group Ho.1) 27 H. ovalis Leam Yong Lum, Trang, Thailand CK 15-5 KP408238 † (Group Ho.1) 28 H. ovalis Leam Yong Lum, Trang, Thailand CK 15-7 KP408239 † (Group Ho.1) 29 H. ovalis Leam Yong Lum, Trang, Thailand CK 19-5 KP408240 † (Group Ho.1) 30 H. ovalis Koh Tan, Suratthani, Thailand CK 24-1 KP408241 † (Group Ho.1) 31 H. ovalis Koh Tan, Suratthani, Thailand CK 24-2 KP408242 † (Group Ho.1) 32 H. ovalis Koh Tan, Suratthani, Thailand CK 24-3 KP408243 † (Group Ho.1) 33 H. ovalis Koh Tan, Suratthani, Thailand CK 24-3.4 KP408244 † (Group Ho.1) 34 H. ovalis Koh Tan, Suratthani, Thailand CK 24-4 KP408245 † (Group Ho.1) 35 H. ovalis Koh Tan, Suratthani, Thailand CK 24-5 KP408246 † (Group Ho.1) 36 H. ovalis Ban Pak Meang, Trang, Thailand PT 46.1.2 KP408247 † (Group Ho.1) 37 H. ovalis Ban Pak Meang, Trang, Thailand PT 46.1.3 KP408248 † (Group Ho.1) 38 H. ovalis Ban Pak Meang, Trang, Thailand PT 46.2.4 KP408249 † (Group Ho.1) 39 H. ovalis Ban Pak Meang, Trang, Thailand PT 46.3.1 KP408250 † (Group Ho.1) 40 H. ovalis Ban Pak Meang, Trang, Thailand PT 47.2.2 KP408251 † (Group Ho.1) 41 H. ovalis Ban Pak Meang, Trang, Thailand PT 47.1.3 KP408252 † (Group Ho.1) 42 H. ovalis Ban Pak Meang, Trang, Thailand PT 62.1 KP408253 † (Group Ho.1) 43 H. ovalis Koh Lidee, Satun, Thailand PT 57.1.1 KP408254 † (Group Ho.1) 44 H. ovalis Leam Yong Lum, Trang, Thailand PT 49.1.5* KP408255 † (Group Ho.2) 45 H. major Trang, Thailand AB436927 12 46 H. major Okinawa, Japan AB243967 23 47 H. major Sumbawa, Indonesia AB436926 12 48 H. major Bali, Indonesia AB436928 12 49 H. major Nha Trang, Vietnam KC175910 20 50 H. major Gyeiktan, Myanmar KF620352 20 51 H. major Mabul Island, Malaysia KF620340 20 52 H. major Leam Yong Lum, Trang, Thailand PT 48.1.1* KP408256 † (Group Hm.2) 53 H. major Leam Yong Lum, Trang, Thailand PT 48.1.2* KP408257 † (Group Hm.3) 54 H. major Leam Yong Lum, Trang, Thailand PT 48.2.3 KP408258 † (Group Hm.1) 55 H. major Leam Yong Lum, Trang, Thailand PT 48.3.1* KP408259 † (Group Hm.1) 56 H. major Leam Yong Lum, Trang, Thailand PT 51.1.2* KP408260 † (Group Hm.1) 57 H. major Koh Libong, Trang, Thailand PT 133.10 KP408261 † (Group Hm.1) 58 H. major Koh Muk, Trang, Thailand CK 16-3 KP408262 † (Group Hm.1) 59 H. major Ko Lao Liang, Trang, Thailand SP 348 KP408263 † (Group Hm.1) 60 H. major Ao Nang, Krabi, Thailand AD 178 a KP408264 † (Group Hm.1) 61 H. major Ao Nang, Krabi, Thailand AD 178 b KP408265 † (Group Hm.1) 62 H. australis South-Western Australia AF366414 29 63 H. hawaiiana Hawaii, USA AF366426 29 64 H. johnsonii Florida, USA AF366425 29 65 H. mikii Kagoshima Pref., Japan AB436929 12 66 H. minor Guam AF366405 29 67 H. minor Philippines AF366406 29 68 H. stipulacea Sicily, Italy AF366436 29 69 H. decipiens Malaysia AF366412 29 * measurement samples in this study † this study

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into the 4 sections and examined as above. The homoscedasticity of data was tested using Levene’s test; and Two-ways ANOVA was employed to com- pare the difference in those characters with respect to species at each age; and Welch ANOVA was employed if data are heteroscedasticity.

RESULTS Molecular phylogeny and ecological aspects The phylogenetic tree obtained with the Ml method is presented in Fig. 4. Both maximum likelihood Fig. 2 Leaf characters of Halophila major that were (Ml) and Bayesian Inference trees have the same measured in each section. LL: leaf length; LW: leaf width; topology. Thirty eight ITS sequences were divided AG: angle between cross veins and mid veins; CVB: cross into two clades consisting of clade I and clade II veins branching; SC: space between cross veins; SM: with 99 bootstrap percentages and 1 of Bayesian distance between intramarginal veins and lamina margin. Inference posterior probabilities. In clade I, both Scale bar is 5 mm. haplotype Ho.1 and Ho.2 were grouped with known iii iv sequences, H. ovalis from GenBank. The 27 samples (Ho.1) were identical but different by 2 bp from H. ovalis AB243973 (JP). The haplotype Ho.2 was ii identical with H. ovalis sequences KF620345(TH), KF620347(TH), AB436938(TH), KF620337(HK), i KF620338(ML2) and KF620339(ML3) but differ- ent by 2 bp from haplotype Ho.1. In clade II, haplotype Hm.1, Hm.2 and Hm.3 were clustered with known sequences, H. major from GenBank. Haplotype Hm.1 (8 samples) were identical with H. major AB436927(TH). Haplotype Hm.2 and Hm.3 were grouped with AB436928 and AB436926 Fig. 3 Leaf age classes. (i) Young or new leaf without from Indonesia, respectively. Hm.1 differed by the petiole; (ii) young leaf nearly mature and shot petiole 5 bp from Hm.2 and 6 bp from Hm.3, while Hm.2 length (2nd of leaf pairs); (iii) fully youngest mature (3rd and Hm.3 differed only 1 bp. The results showed leaf pairs); (iv) old leaf (4th–5th leaf pairs). Scale bar is the nucleotide differences among individuals of 3 cm. H. ovalis clade and H. major clade were 0–15 nu- cleotides and 0–14 nucleotides, respectively. Intra- vein branching counted from the base of cross vein species variations were 0–0.014% (H. major), 0– which branched in the end of cross vein. The space 0.015% (H. ovalis) and 0–0.018% (H. nipponica). between cross veins (SC) and the space between Inter-species variations between H. major/H. ovalis, the intra-marginal vein and the lamina margin (SM) H. major/H. nipponica or H. ovalis/H. nipponica measured as the distance between each characters. were 0.04–0.057%, 0.05–0.068% or 0.031–0.047%, The angle of cross veins ascending measured at the respectively. angle between the mid vein and the cross vein in H. ovalis has occurred in both Andaman Sea each section. All these character parameters were and Gulf of Thailand while H. major was found examined using an image analysis program (imageJ only in the Andaman Sea. H. ovalis and H. major software version 1.46r). Three replicates of each from In Leam Yong Lum, Trang province showed character parameters were made. The leaves were great genetic variations, covered both haplotypes of also divided into 4 age classes (Fig. 3): Age (i) is the H. ovalis (Ho.1 and Ho.2) and all of H. major (Hm.1, young or new apical leaf with petiole development, Hm.2, and Hm.3). age (ii) is young leaf nearly mature and with a short petiole (2nd leaf pairs), age (iii) is fully young Morphological observations mature (3rd leaf pairs), and age (iv) is an old leaf The molecular analysis revealed that the large- (4th leaf pairs). Leaves at each age were divided leafed Halophila species is H. major (Zoll.) Miq. www.scienceasia.org ScienceAsia 41 (2015) 83

AB243973_H. ovalis_JP CK13-1, CK14-2, CK15-5, CK19-5, CK24-1, CK24-2, PT45.1.1, PT52.1.2, ... n = 27_TH Ho.1 AB243975_H. ovalis_JP KC175909_H. ovalis_VN KF620350_H. ovalis_TH AB436939_H. ovalis_TH 84/1 KF620346_H. ovalis_ML1 KF620355_H. ovalis_INDIA2 60/1 84/1 KF620353_INDIA1 PT 49.1.5_TH Ho.2 H. ovalis KF620345_H. ovalis_TH KF620347_H. ovalis_TH 93/1 KF620377_H. ovalis_HK KF620338_H. ovalis_ML2 KF620339_H. ovalis_ML3 AB436938_H. ovalis_TH 78/- AF366431_H. ovalis_AUS 88/1 AF366426_H. ovalis[H. hawaiiana TYPE]_HW AB436940_H. ovalis[H. minorTYPE]_INDO AB436930_H. ovalis_INDO AF366425_H. ovalis[H. minor TYPE]_INDO AF366405_H. minor_GM 99/1 100/1 AF366406_H.minor_PHI

PT48.1.2_TH Hm.3 73/1 62/1 AB436926_H. majorTYPE_INDO PT48.1.1_TH Hm.2 AB436928_H. major_INDO 80/1 KC175910_H. major_VN KF620340_H. major_ML H. major AF366414_H. major[H. australis]_AUS AB436929_H. major[H. mikiiTYPE]_JP AB243967_H. major[H. euphlebia]_JP AB436927_H. major_TH 100/1 PT51.1.2,PT348,AD178A, ... , n = 8_TH Hm.1 KF620352_H. major_MYN AF366412_H. decipiens_ML AF366436_H. stipulacea_IT

0.2 Fig. 4 ML tree of Halophila species (nrITS: 635 bp). ML bootstrap values (> 50) and BI posterior probabilities (> 0.80) are indicated at nodes.

A total of 20 morphological leaf characters in 4 character between H. major and H. ovalis were age classes were examined and compared between closely observed as also summarized in Table 2. Leaf H. major and H. ovalis. The leaf characters showed length and width ranged 23.9–29.4 mm and 10.8– the variation between the species among age classes 12.6 mm in H. major and 10–17 mm and 4.3– (Table 2). Out of the 20 examined characters, there 836 mm in H. ovalis, respectively, (Fig. 5a–d). The were 9 morphological leaf characters that featured numbers of cross veins were 14–18 veins in H. major significant differences between species in each age; and were 9–16 veins in H. ovalis (Fig. 5e). Space which were leaf length, leaf width in each section, between cross veins at 50%, 75% and 100% leaf number of cross veins, space between cross veins area were slightly increased in space with aged. (SC) in each section (except at 25% leaf area), space However, H. major had wider space between cross between the intra-marginal vein and lamina margin veins than H. ovalis (Fig. 5f–h). Interestingly, the (SM) at the leaf tip and ratio between ½ leaf width ratio between ½ leaf width and the space between and the space between the intra-marginal vein and the intra-marginal vein and lamina margin was lamina margin (Fig. 5a–i). The differences in leaf clearly different. A much greater ratio was found

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Table 2 Summary of morphological characters between H. major and H. ovalis populations.

† * Characters LL LW CV CVB SC SM SMa AG ½ LW/SM (mm) (mm) (no.) (no.) (mm) (mm) (mm) (°) H. ovalis (n) ‡ # age (i) (8) 6.8–10.1 4.6–5.6 7–8 (2–3) n/d 0.1–0.4 0.37–0.39 59.3–77.3 1:8–9.2 age‡(ii) (7) 12.7–18.2 6.1–8.2 11–14 (1–4) 0.4–1.3 0.2–1.2 0.3–0.5 36.3–89.4 1:3.6–12.7 age‡(iii) (11) 11.2–17.0 4.3–8.3 9–16 (0–3) 0.4–1.4 0.2–1.1 0.3–0.5 30.4–88.5 1:4.1–9.7 age‡(iv) (12) 10.0–15.0 4.6–7.6 9–14 (0–3) 0.3–1.1 0.2–0.6 0.3–0.5 43.4–79.4 1:6.8–12.1 19 Japan 12–18 4–8 12–16 n/d 0.8–1.1 0.25–0.4 n/d n/d 1:10–16 24 Vietnam 9–12 3.7–7.0 8–16 n/d n/d 0.3 n/d 45–80 1:9–17 H. major (n) age‡(i) (9) 11.7–19.5 7.1–12.6 9–16 (0–5) 0.5–1.5 0.1–0.5 0.2–0.5 36.5–87.4 1:16.6–22.2 age‡(ii) (5) 13.0–25.7 5.6–14.8 12–18 (1–6) 0.5–1.5 0.1–1.5 0.2–0.5 30.1–77.7 1:18.5–19.9 age‡(iii) (15) 25.9–29.4 10.8–12.6 14–18 (1–6) 0.6–2.0 0.1–0.6 0.4–0.6 27.7–75.4 1:16.6–27.1 age‡(iv) (8) 23.9–27.4 10.8–12.3 15–18 (1–6) 0.6–1.5 0.2–0.6 0.4–0.6 36.9–67.3 1:17.2–27.7 19 Japan 10–25 9–11 18–22 n/d 0.7–1.25 0.16–0.5 n/d n/d 1:20–25 23 Japan 10–30 5–15 12–19 n/d 0.7–1.25 0.1–0.2 n/d n/d 1:20–25 24 Vietnam 10–18 9–12 16–22 n/d n/d 0.2–0.25 n/d 60–80 1:24–25 † LL: leaf length; LW: Leaf width; CV: cross veins; CVB: cross veins branching; SC: space between cross veins; SM: space between intra-marginal veins and leaf margin; SMa: space between intra-marginal veins and leaf margin at apex of leaf; AG: cross veins angle; ½ LW/SM: Half of leaf width per space between intra-marginal veins and leaf margin. ‡ Populations in this study from Andaman Sea, Thailand. * Cross veins branching is common for all populations except for H. ovalis from Vietnam 24. # n/d = no data. in H. major and ranged between 1:16.58–27.74 and shape and shows some overlap in size among leaf 1:3.59–12.65 in H. ovalis without any overlapping age groups (Table 2), they can be clearly distin- values (Fig. 5i). This, in fact, could be a dependable guished by (1) a significant larger leaf size in all character for the identification of H. major. leaf ages, especially in the age (iii) and (iv); and (2) The ½ ratio between leaf width and the space DISCUSSION between the intra-marginal vein and lamina margin H. major is reported to be distributed in Western Pa- is significantly higher in H. major than in H. ovalis. cific region including Vietnam, Indonesia, Malaysia Thus we suggest that leaf ages as well as the ½ and the Indian Ocean including Thailand and Myan- ratio are important for distinguishing these 2 closely mar either through the morphological or molecular similar species; and as well as other Halophila spp. information 12, 19, 20. This study was the first re- Although there are significantly more cross port, which combined both morphological features veins in H. major in most mature leaves, the num- and molecular information to identify H. major ber of cross veins in this study ranged only 14– and suggested some key characters to identify this 18, which is much lower than those reported from closely similar species. Unlike the study of Nguyen Japanese populations (18–22 cross veins) 19. This et al 20, where high genetic diversity of H. ovalis suggested that there is high variability in this char- was reported across the Indo-Pacific Ocean, our acter in Halophila spp. between various populations results showed the identical sequences of H. major (Table 2). Overlap of characters is common and or between Trang and Krabi provinces (haplotype conspecific within this group, e.g., H. minor and Hm.1, Fig. 4), however a much smaller scale. The H. ovalis, H. major and H. miki, H. nipponica, H. ok- distance between those sites are less than 100 km, inawensis and H. gaudichaudii 23. The nomenclature where both influenced by the same water current of the Halophila group is still confounding which thus low genetic diversity was expected. It would limits the risk assessment of the dangers to the be interesting to further understand, dispersal, re- world’s seagrass species 29. In addition to the distin- cruitment and sexual reproduction of this species. guishing morphological and ITS characters, sexual Although H. major and H. ovalis have similar reproductive features, flowers and fruits, would www.scienceasia.org ScienceAsia 41 (2015) 85

35 (a) 16 (b) (c) 16 d c,d d d d d 28 c c,d d b,c 12 b,c 12 b 21 b,c b b,c a,b a,b a,b mm b a,b mm 8 a,b

mm 8 14 a a a 4 4 7

0 0 0 Hm-1 Hm-2 Hm-3 Hm-4 Ho-1 Ho-2 Ho-3 Ho-4 Hm-1 Hm-2 Hm-3 Hm-4 Ho-1 Ho-2 Ho-3 Ho-4 Hm-1 Hm-2 Hm-3 Hm-4 Ho-1 Ho-2 Ho-3 Ho-4 16 (d) (e) (f) 20 c,d d d c,d d 1.8 d b,c b d 12 b,c 15 b b b,c,d c,d a,b,c a,b,c 1.2 a,b 8 a,b a

mm a,b a,b 10

veins a mm

a 0.6 4 5

* 0 0 0.0 Hm-1 Hm-2 Hm-3 Hm-4 Ho-1 Ho-2 Ho-3 Ho-4 Hm-1 Hm-2 Hm-3 Hm-4 Ho-1 Ho-2 Ho-3 Ho-4 Hm-1 Hm-2 Hm-3 Hm-4 Ho-1 Ho-2 Ho-3 Ho-4 1.8 1.8 (g) (h) 35 (i) c b a 28 c a a b a,b b,c a a,b a a,b a,b b 1.2 1.2 a a,b a 21 mm mm 14 a a a 0.6 0.6 a 7 * * 0.0 0.0 0 Hm-1 Hm-2 Hm-3 Hm-4 Ho-1 Ho-2 Ho-3 Ho-4 Hm-1 Hm-2 Hm-3 Hm-4 Ho-1 Ho-2 Ho-3 Ho-4 Hm-1 Hm-2 Hm-3 Hm-4 Ho-1 Ho-2 Ho-3 Ho-4 Fig. 5 Summary of morphological characters between H. major (Hm-) and H. ovalis (Ho-) populations among 4 age classes. Medians are highlighted in bold; bars represent the 25% and 75% quartiles; whiskers represent the lowest and highest data points. (a) Leaf length (Welch = 145.99, p-value = 0.000); (b) leaf width at 25% leaf area (Welch = 90.46, p-value = 0.000); (c) leaf width at the 50% leaf area (Welch = 183.19, p-value = 0.000); (d) leaf width at 75% leaf area (Welch = 597.11, p-value = 0.000); (e) number of cross veins (Welch = 44.69, p-value = 0.000); (f) space between cross veins at 50% leaf area (Welch = 11.09, p-value = 0.000) and * = no data; (g) space between cross at 75% leaf area (Welch = 12.34; p-value = 0.000); (h) space between cross at 100% leaf area (Welch = 5.52, p-value = 0.000) and * = no data; (i) ratio between 0.5 leaf width and space between intra marginal vein and leaf edge (Welch = 77.52, p-value = 0.000). complete the description of the species. Establishing 4. Short FT, Moore GE, Peyton KA (2010) Halophila those characters was outside the realm of this study. ovalis in the Tropical Atlantic Ocean. Aquat Bot 93, 141–6. Acknowledgements: This study was supported by 5. Ralph PJ, Burchett MD (1995) Photosynthetic re- Higher Education Research Promotion and National Re- sponses of the seagrass Halophila ovalis (R. Br.) Hook. f. to high irradiance stress, using chlorophyll a search University Project of Thailand, Office of the Higher fluorescence. Aquat Bot 51, 55–66. Education Commission (NRU). Prof. Larry B Liddle has 6. Ralph PJ (1998) Photosynthetic response of labora- improved the English of this manuscript. AP and SS tory-cultured Halophila ovalis to thermal stress. Mar would like to thank National Research Council of Thailand Ecol Progr 171, 123–30. (NRCT)-Japan Society for the Promote of Science (JSPS) 7. Shafer DJ, Sherman TD, Wyllie-Echeverria S (2007) research collaboration scheme. Do desiccation tolerances control the vertical distri- bution of intertidal seagrasses? Aquat Bot 87, 161–6. 8. Tanaka Y, Nakaoka M (2004) Emergence stress and REFERENCES morphological constraints affect the species distribu- 1. den Hartog C (1970) Seagrasses of the World, tion and growth of subtropical intertidal seagrasses. North Holland Publishing Company, Amsterdam, The Mar Ecol Progr 284, 117–31. Netherlands. 9. Kuo J, den Hartog C (2001) Seagrass 2. Short F,Carruthers T, Dennison W,Waycott M (2007) and identification key. In: Short FT, Coles RG (eds) Global seagrass distribution and diversity: A biore- Global Seagrass Research Methods, Elsevier Science gional model. J Exp Mar Biol Ecol 350, 3–20. B.V.,Amsterdam, pp 31–58. 3. Larkum AWD, Orth RJ, Duarte CM (2006) Sea- 10. Chansang H, Poovachiranon S (1994) The distribu- grasses: Biology, Ecology, and Conservation, Springer, tion and species composition of seagrass beds along Dordrecht, The Netherlands. the Andaman Sea coast of Thailand. Phuket Mar Biol

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